Potentiation of immunogenic response

ABSTRACT

An enhanced immune response to antigens, particularly normally weakly-immunogenic viral antigens, such as the HA antigen from influenza virus, is achieved by coadministering the antigen in two different physio-chemical forms, particularly to enable presentation of antigen both by B cells and accessory cells.

This application is a continuation of application Ser. No. 07/943,173,filed Sep. 14, 1992 now abandoned.

FIELD OF THE INVENTION

The present invention relates to vaccination and, in particular, toformulating vaccines so as to achieve an enhanced immunogenic responseto an antigen.

BACKGROUND OF THE INVENTION

Vaccination is a procedure whereby an immune response to an antigen canbe achieved to protect a host from infection. Some antigens elicit astrong immune response and some a weak response. Attempts have been madeto enhance the immune response of weakly-immunogenic materials. The useof chemical adjuvants achieves such potentiation but generally suchmaterials are toxic chemicals which cannot be used in humans.

Another procedure for achieving potentiation is to conjugate theweakly-immunogenic material to a strongly-immunogenic material andadminister the conjugate in a vaccine. For example, a conjugate of thecapsular polysaccharide of Haemophilus influenzae type b to diphtheriatoxoid, as described in U.S. Pat. Nos. 4,496,538 and 4,619,828, or aconjugate of a weak antigen to a monoclonal antibody targetingantigen-presenting cells, as described in U.S. Pat. No. 4,950,480, maybe employed.

SUMMARY OF INVENTION

In accordance with the present invention, there is provided a novelprocedure of vaccination to elicit an enhanced antibody response to anantigen in a naive animal by administering the antigen in at least twodifferent physio-chemical forms. The two different physio-chemical formsof the same antigen are administered simultaneously in a naive animal toachieve the greatest degree of potentiation and may be administered at asingle or two injection sites.

In order for the enhanced immune response to be achieved, it isnecessary that the animal to which the antigen is coadministered,including humans, be naive, i.e. the animal has not been previously beenimmunized by a highly-immunogenic form of the antigen. Co-administrationof the antigen to a primed animal elicits no enhancement of immuneresponse.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 and 2 contain graphical data of HAI titers achieved by variousforms of HA antigen in naive guinea pigs, as detailed in Example 1below;

FIGS. 3 to 5 contain graphical data of IgG anti-HA responses achieved byvarious forms of HA antigen in guinea pigs, as detailed in Example 2below;

FIGS. 6 and 7 contain graphical data of HAI titers achieved by variousforms of HA antigen in primed guinea pigs, as detailed in Example 3below; and

FIGS. 8 and 9 contain graphical data of the immune response achieved byvarious forms of OspA in naive mice, as detailed in Example 4 below.

THEORY

While the applicant does not wish to be bound to any specific theory toexplain why the present invention is effective in achieving potentiationof an antigenic response by the simultaneous administration of at leasttwo different physio-chemical forms of the antigen, the followingtheoretical explanation can be made.

It is known that both B cells and accessory cells must present antigensto T-cells to initiate an antibody response to the antigen in naiveanimals (refs. 1, 2, 3, 4 and 5). B cells and accessory cells may havepreferences for different physio-chemical forms of antigens. Thesepreferences may result from B cells and accessory cells using differentmechanisms for internalizing antigens.

B cells internalize antigens via specific binding to their cell surfaceimmunoglobulins (refs. 6, 7) and can present soluble antigens inconcentrations as low as 1 ng/ml (ref. 8). Accessory cells, which aremacrophages and dendritic cells, on the other hand, internalize antigensby non-specific phagocytizing and pinocytizing the antigens (ref. 9).Macrophages can present soluble antigens if they are at concentrationsof approximately 100 μg/ml (ref. 8). Other research (ref. 10) hasdemonstrated that the concentration of soluble antigen needed bymacrophages can be decreased by binding the antigen to a particulatestructure.

During the generation of an antibody response, B cells and accessorycells present antigens to T-cells at two different stages of T cellactivation/differentiation. Research has demonstrated that naive T cellsmust first interact with antigen presenting accessory cells to becomeactivated helper T cells (refs. 11, 12, 13, 14). The inventors believethat particulate forms of antigens, as employed herein, effectivelymediate the accessory cell activation of naive T cells. Thisinteraction, however, is insufficient to induce the B cells to respondto a T cell-dependent antigen. Direct cell-to-cell contact between Bcells and activated helper T cells is required for the induction ofantibody secretion from B cells (ref. 15). This interaction is mediatedby B cells processing and presenting the antigen to activated T cell(ref. 16). This type of B cell-T cell interaction is termed cognate Tcell help. The inventors believe that a soluble form of antigen employedherein best mediates B cells interacting with helper T cells.

The order of the two interactions is essential to bring about therequired immune response in naive T cells. It has recently been shownthat antigen presentation of B cells to naive T cells induces T celltolerance rather than activation (refs. 2, 3). Since optimal immuneresponses require efficient antigen presentation by both B cell andaccessory cells, such optimal response can be achieved in the presentinvention by simultaneously administering the antigen in twophysio-chemical forms at either one or two sites of injection.

GENERAL DESCRIPTION OF INVENTION

As stated above, the novel method of achieving potentiated immuneresponse in a naive animal, including humans, to an antigen is toadminister the antigen simultaneously in at least two differentphysio-chemical forms. The invention is broadly applicable to a widevariety of antigens, particularly viral, bacterial, fungal, protozoanand parasite protein, and is particularly useful with respect toantigens containing protective epitopes that normally exhibit a weakimmunogenic response.

Among the viral antigens to which the invention may be applied are thegp120 and gp160 proteins of retroviruses especially HIV, thehaemagglutinin antigen of influenza and other viral proteins associatedwith viral membranes.

The invention is illustrated hereinafter with respect to thehaemagglutinin antigen (HA) from influenza virus but it will be apparentfrom the results given for the HA antigen and the discussion above thatthe invention has application to a wide range of antigens. Alsopresented below are data with respect to the immune response to theouter surface protein A (OspA) of the B. burgdorferi spirochete (i.e. abacterial protein) in different physio-chemical forms. Lipidate OspA isa strong immunogen and hence coadministration with other forms of theOspA generally is not required. However, the results presented show thegenerality of the procedure.

One particular viral protein to which the invention may be applied isthe gp120 protein of human immunodeficiency virus (HIV). The gp120protein of HIV contains protective epitopes but is a poor immunogen. Theimmune response to gp120 can be potentiated by coadministering gp120protein with inactivated HIV virions, gp160 or pseudovirions. The gp160protein is the precursor protein that is proteolytically cleaved to formgp120 and gp40. The gp120 protein normally is associated with HIVvirions via gp40. Purified gp120 protein is a soluble protein which ispoorly immunogenic while viral particulate and gp160 protein are moreimmunogenic. Coadministration in accordance with the present inventionmay achieve an enhanced immune response to the gp120 protein.

The different physio-chemical forms of the antigen for coadministrationmay vary widely, depending on the antigen chosen and the specificantigenic forms of the antigen which might be available. Preferably, thetwo forms are tailored to provide for antigen presentation both by Bcells and by accessory cells to T-cells to initiate antibody response.

For example, one physio-chemical form may be soluble while the other maybe insoluble and/or particulate, as in the case of HA antigen.Alternatively, the different physio-chemical forms of the antigen may bea lipidated protein and a non-lipidated protein, as in the case of OspAantigen. In addition, the different physio-chemical forms of the antigenmay comprise proteins with and without hydrophobic region. Further, thedifferent physio-chemical forms of the antigen may comprise proteinswhich have been engineered, for example, by genetic engineering orchemical synthesis, to be provided with or without specific epitopes orregions.

EXAMPLES EXAMPLE 1

This Example demonstrates the effect of coadministration of differentphysio-chemical forms of the HA antigen from influenza virus.

Several different physio-chemical forms of HA exist, namely HA(p), splitHA and inactivated whole virus. HA(p) is a highly purified form of HAthat has had its hydrophobic tail removed by bromelain cleavage and issoluble in water. Split HA is a detergent extracted and partiallypurified form of the HA antigen. Inactivated whole virus is formalininactivated whole virus particles.

Split HA and inactivated whole virus are immunogenic in naive animalsand humans. HA(p) is not immunogenic in naive animals or infants, eventhough it is antigenic in antibody-antigen reactions.

There was conducted two series of experiments in which guinea pigs wereimmunized with various physio-chemical forms of HA from the A/Taiwaninfluenza strain, alone or in combination, and their responses weremeasured by haemagglutination inhibition (HAI) titers, HAI titers beingknown to correlate wall with protective immune responses. The resultsobtained in the experiments were plotted graphically and appear as FIGS.1 and 2.

In these experiments, the amount of HA(p) was maintained constant (1.0μg) and the amount of added whole inactivated virus was varied. Of thethree amounts of whole inactivated virus employed (1.0 μg, 0.1 μg and0.01 μg), immune responses were best potentiated by co-administrationusing 0.1 μg whole inactivated virus, as may be seen from FIGS. 1 and 2.

When the titers for this combination were compared to the titers forHA(p) or 0.1 μg whole inactivated virus alone, coadministrationpotentiated immune responses four to seven fold at two to four weeksafter the boost. At the higher dose of 1.0 μg of whole inactivatedvirus, immune responses to coadministration were equal to the responsesto the virus alone, again as seen in FIGS. 1 and 2. At the low dose of0.01 μg whole inactivated virus, the immune response to bothcoadministration and whole inactivated virus alone were low (see FIG.2). Since HAI titers correlate well with protective immune responses,these results suggest that coadministration enhances protective immuneresponses in guinea pigs.

The co-administration of split HA and HA(p) also enhanced anti-HAantibody responses in guinea pigs. Maximal enhancement bycoadministration was observed using 0.1 μg of HA(p) and 0.1 μg of splitHA, as may be seen from the results of FIGS. 1 and 4. A three- toseven-fold enhancement in HAI titers was observed using these amounts ofantigen.

EXAMPLE 2

In addition to the results obtained in Example 1, antibody responseswere analyzed by EIA (ELISA immunoassay) to determine whether theenhancement of HAI titers by coadministration was related to the totalamount of IgG anti-HA antibody generated. In these experiments, HA-e (ahighly-purified form of HA that retains its hydrophobic tail) was usedto coat the wells of the EIA plate and anti-guinea pig IgG was used as adetecting antibody. The dilution curves of experimental antisera werecompared to the dilution curve of a standard guinea pig anti-serum and,on the basis of that comparison, the units of IgG anti-HA werecalculated in each sera.

Using the same guinea pig sera, a good correlation was found when theresults of the EIA, as seen in FIG. 3, were compared with the results ofthe HAI, as seen in FIG. 2. These results show that co-administration ofthe HA in different forms enhances the total amount of IgG generatedagainst HA.

The results of EIA on sera from an experiment using split HA, as seen inFIG. 5, indicated that the increased HAI titers from co-administrationwere the result of increased amounts of anti-HA antibodies. From theresults set forth in Examples 1 and 2, it is apparent that the levels ofantibody generated to coadministration with split HA generally were lessthan those to coadministration with whole inactivated virus, as may beseen from FIG. 1 and a comparison of FIGS. 2 and 4 and FIGS. 3 and 5.

In the experiments reported in Examples 1 and 2, naive animals were usedto evaluate coadministration.

EXAMPLE 3

This example illustrates the effect of coadministration of HA in primedanimals.

Guinea pigs were primed with either 1.0 μg of whole inactivated virus(results depicted in FIG. 6) or 1.0 μg of split HA (results depicted inFIG. 7). Three weeks later, the guinea pigs were given secondaryimmunization of either single flu antigen or coadministered fluantigens. The results shown in FIGS. 6 and 7 indicate thatco-administration does not enhance anti-HA results in primed animals andhence the co-administration technique is useful only in naive animals,if an enhanced immune response is to be achieved.

These results also show that the superior antigen for recalling memoryresponses was HA(p) alone, while immunization with HA(p) at the primaryand secondary immunization did not generate a significant immuneresponse. These results show that HA(p) can recall memory immuneresponses to the HA antigen but cannot itself generate memory. The useof the weakly-immunogenic HA(p) to achieve an enhanced secondary immuneresponse from a HA primed animal forms the subject of copending U.S.patent application Ser. No. 943,247 filed Sep. 14, 1992 by Becker et aland assigned to the assignee hereof.

EXAMPLE 4

This Example demonstrates the effect of different physio-chemical formsof the OspA protein of B. burgdorferi spirochete.

OspA lipoprotein (OspA-L) is a very potent immunogen. Removal of thelipid moiety from OspA dramatically decreases its immunogenicity but notits antigenicity, as described in copending U.S. patent application Ser.No. 888,765 filed May 27, 1992, assigned to the assignee hereof and thedisclosure of which is incorporated herein by reference.

A small dose of OspA-L was coadministered to C3H/He mice with a largedose of OspA-NL and the response compared to the responses of OspA-L orOspA-NL alone. The mice were immunized at days 0 and 21 with theantigens and the mice were bled on day 35. The dilution curves of anELISA assay of sera from the mice were plotted graphically and theresults are shown in FIG. 8. Immune responses also are shown in FIG. 9.

As may be seen from this data, a potentiation of OspA response wasachieved by coadministration of OspA-L and Ospa-NL relative toadministration of OspA-L or OspA-NL alone.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a novelmethod of obtaining an enhanced immune response to a viral antigen bycoadministering the antigen in different physio-chemical forms.Modifications are possible within the scope of this invention.

REFERENCES

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2. "Antigen Presentation in Acquired Immunological Tolerance", Parker etal, The FASEB Journal, Vol. 5, October 1991, pp. 2771-2784.

3. "Do Small B Cells Induce Tolerance", Eynon et al, TransplantationProceedings, Vol. 23, No 1 (February) 1991: pp. 729-730.

4. "Small B Cells as Antigen-Presenting Cells in the Induction ofTolerance to Soluble Protein Antigens" by Eynon et al, J. Exp. Med. Vol.175, January 1992, pp. 131-138.

5. "Role of B Cell Antigen Processing and Presentation in the HumoralImmune Response", Myers, The FASEB Journal, Vol. 5, August 1991, pp.2547-2553.

6. "Antigen Presentation by Hapten-Specific B Lymphocytes", Abbas et al,J. Immun. Vol. 135, No. 3, September 1985, pp. 1661-1667.

7. "Requirements for the Processing of Antigen by Antigen-Presenting Bcells", Grey et al, J. Immun., Vol. 129, No. 6, Dec. 1982, pp.2389-2395.

8. "Antigen-Specific B Cells Efficiently Present Low Doses of Antigenfor Induction of T Cell Proliferation", Malynn et al, J. Immun. Vol.135, No. 2, August 1985, pp. 980-987.

9. "Antigen-Presenting Function of the Macrophage", Unanue, Ann. Rev.Immunol., 1985, 2: 395-428.

10. "Analysis of TX Lymphocyte Reactivity to Complex Antigen Mixtures bythe Use of Proteins coupled to Latex Beads", Wirbelauer et al, Immun.Letters, 23 (1989/1990), 257-262.

11. "The Function and Interrelationships of T. Cell Receptors, Ir Genesand other Histocompatibility Gene Products", Katz et al, Transplant.Rev. (1975), Vol. 22, pp. 175-195.

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14. "The Role of H-2-Linked Genes in Helper T-Cell Function", Swierkoszet al, J. Exp. Med., 1978, Vol. 147, pp. 554-570.

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What we claim is:
 1. A method for achieving an enhanced immune responseto an antigen in an animal which is a naive animal, wherein a naiveanimal is an animal which has not been previously immunized by a highlyimmunogenic form of the antigen, which method comprises the stepsof:simultaneously administering the antigen to the naive animal in atleast two different physiochemical forms to provide a synergistic immuneresponse to the antigen in the naive animal greater than the immuneresponse to the individual physiochemical forms of the antigen in thenaive animal, wherein said antigen is a bacterial protein.
 2. The methodof claim 1 wherein one of the physiochemical forms favors presentationof the antigen by B cells to T cells in the naive animal and the otherof the physiochemical forms favors presentation of the antigen byaccessory cells to T cells in the naive animal.
 3. The method of claim 2wherein one physiochemical form of the antigen is soluble while theother is insoluble and/or particulate.
 4. The method of claim 2 whereinone physiochemical form of the antigen is lipidated and the otherphysiochemical form is non-lipidated.
 5. The method of claim 4 whereinthe antigen is OspA protein from Borrelia burgdorferi and the differentphysiochemical forms of the antigen comprise OspA-L and OspA-NL.
 6. Themethod of claim 2 wherein one physiochemical form of the antigen is aprotein having a hydrophobic region and the other physiochemical form isthe protein lacking the hydrophobic region.
 7. The method of claim 2wherein one physiochemical form of the antigen is a protein engineeredto contain a specific epitope and/or region and the other is the proteinlacking such specific epitope and/or region.
 8. The method of claim 1wherein the animal is a human.
 9. A vaccine for eliciting an immuneresponse to an antigen in an animal which is a naive animal, wherein anaive animal is an animal which has not been previously immunized by ahighly-immunogenic form of the antigen, including humans, which vaccinecomprises:a first physiochemical form of the antigen favoringpresentation of the antigen by B cells to T cells in the animal, asecond physiochemical form of the antigen favoring presentation of theantigen by accessory cells to T cells in the animal, and aphysiologically-acceptable carrier for the first and secondphysiochemical forms of the antigen, whereby a synergistic enhancedimmune response to the antigen is achieved upon administration of thevaccine to the naive animal in comparison to either of thephysiochemical forms administered alone, wherein the antigen is abacterial protein.
 10. The vaccine of claim 9 wherein the firstphysiochemical form of the antigen is soluble while the other isinsoluble and/or particulate.
 11. The vaccine of claim 9 wherein onephysiochemical form of the antigen is lipidated and the otherphysiochemical form is not lipidated.
 12. The vaccine of claim 11wherein the antigen is the OspA protein from Borrelia burgdorferi andthe different physiochemical forms of the antigen comprise OspA-L andOsp-NL.
 13. The vaccine of claim 9 wherein one physiochemical form ofthe antigen is a protein having a hydrophobic region and the other isthe protein lacking the hydrophobic region.
 14. The vaccine of claim 9wherein one physiochemical form of the antigen is a protein engineeredto contain a specific epitope and/or region and the other is the proteinlacking such specific epitopes and/or region.
 15. A method for achievingan enhanced antigenic response to an antigen in an animal which is anaive animal, wherein a naive animal is an animal which has not beenpreviously immunized by a highly immunogenic form of the antigen, whichmethod comprises the steps of:simultaneously administering the antigento the naive animal in at least two different physiochemical forms toprovide a synergistic antigenic response to the antigen in the naiveanimal greater than the antigenic response to the individualphysiochemical forms of the antigen in the naive animal, wherein saidantigen is an influenza virus antigen, and wherein the two differentphysiochemical forms provide for presentation of influenza virus antigenboth by B cells and by accessory cells to T cells to initiate antigenicresponse, and the two forms are from one influenza strain.
 16. A vaccinefor eliciting an antigenic response to an antigen in an animal which isa naive animal, wherein a naive animal is an animal which has not beenpreviously immunized by a highly-immunogenic form of the antigen,including humans, which vaccine comprises:a first physiochemical form ofthe antigen favoring presentation of the antigen by B cells to T cellsin the animal, a second physiochemical form of the antigen favoringpresentation of the antigen by accessory cells to T cells in the animal,and a physiologically-acceptable carrier for the first and secondphysiochemical forms of the antigen, whereby a synergistic enhancedantigenic response to the antigen is achieved upon administration of thevaccine to the naive animal in comparison to either of thephysiochemical forms administered alone, wherein said antigen is aninfluenza virus antigen, and wherein the two different physiochemicalforms provide for presentation of influenza virus antigen both by Bcells and by accessory cells to T cells to initiate antigenic response,and the two forms are from one influenza strain.
 17. A method forachieving an enhanced antigenic response to an antigen in an animalwhich is a naive animal, wherein a naive animal is an animal which hasnot been previously immunized by a highly immunogenic form of theantigen, which method comprises the steps of:simultaneouslyadministering the antigen to the naive animal in at least two differentphysiochemical forms to provide a synergistic antigenic response to theantigen in the naive animal greater than the antigenic response to theindividual physiochemical forms of the antigen in the naive animal,wherein said antigen is an influenza virus antigen, and wherein the twodifferent physiochemical forms comprise a soluble form and an insolubleform of the influenza virus antigen from one influenza strain.
 18. Avaccine for eliciting an antigenic response to an antigen in an animalwhich is a naive animal, wherein a naive animal is an animal which hasnot been previously immunized by a highly-immunogenic form of theantigen, including humans, which vaccine comprises:a firstphysiochemical form of the antigen favoring presentation of the antigenby B cells to T cells in the animal, a second physiochemical form of theantigen favoring presentation of the antigen by accessory cells to Tcells in the animal, and a physiologically-acceptable carrier for thefirst and second physiochemical forms of the antigen, whereby asynergistic enhanced antigenic response to the antigen is achieved uponadministration of the vaccine to the naive animal in comparison toeither of the physiochemical forms administered alone, wherein theantigen is an influenza virus antigen, and wherein the two differentphysiochemical forms comprise a soluble form and an insoluble form ofthe influenza virus antigen from one influenza strain.